International Journal of Agricultural and Food Research
ISSN 1929-0969 | Vol. 3 No. 1, pp. 14-40 (2014)
www.sciencetarget.com
Transgenic Bt-Plants and the Future of Crop Protection
(An Overview)
Reda A. Ibrahim1, 2 and Dalia M. Shawer2, 3*
1
Department of Biology, Faculty of science, Taibah University, Al-Madinah, Saudi Arabia
2
3
Dept. of Economic Entomology, Faculty of Agriculture, Kafrelsheikh University, Egypt
Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University
of Florida, USA
Abstract
One of the best modern agricultural defenses against plant-eating insects is Bacillus thuringiensis (Bt),
which either can be applied to the surface of the plant, to provide temporary protection, or can be
genetically engineered into the plant to protect it against insects throughout its lifespan. Plants can be
genetically engineered to produce their own Bt crystal protein (CP), which is toxic to the pest species of
concern. As the insect feeds on the plant, it ingests the CP and suffers the same fate as if the leaf tissue
was sprayed with Bt. The use of commercial crops expressing Bt toxins has increased in the recent years
due to their advantages in plant protection and lower production costs, however, insects-developed
resistance against plant defense mechanisms and the consequent effects of Bt-plants on non target species
are hence considered disadvantages. This is still a controversial topic and the question is: Within the next
few years, will Bt-plants provide hope for the future of crop protection?
Keywords: Bt-plants, transgenic, plants, crop, protection
Introduction
There are an estimated 67000 pest species that
damage agricultural crops, of which approximately
9000 species are insects and mites (Ross and
Lembi, 1985). Insect-pests are the major cause of
crop losses (Kumar et al., 2008). An average of
15% of crops worldwide is currently lost to insects
(Maxmen, 2013). Insects cause direct losses to the
agricultural crops, in addition to the indirect losses
due to impaired quality of the produce and their
role as vectors of various plant pathogens (Kumar
et al., 2006). In the past, humans have searched for
crop plants that can survive and produce under
different biotic and abiotic stresses. Ancient
farmers searched for pest resistance genes in their
crops, sometimes by actions as simple as collecting
seed from only the highest-yielding plants in their
fields (COMESA, 2007).
Although the development of chemical insecticides
during the last 40 years guaranteed a production
increase in agriculture, contamination of the
environment by pesticides is increasing due to their
usage in crop protection (Oerke, 2006). Using of
pesticides led to contamination of water and food
sources, poisoning of non-target beneficial insects
and development of insect populations resistant to
the chemical insecticides (Kumar et al., 2008;
Matsumura, 1975; Scheyer et al., 2005; Tanabe et
al., 1983). The adverse effect of chemical insect-
* Corresponding author: [email protected]
International Journal of Agricultural and Food Research | Vol. 3 No. 1, pp. 14-40
cides on the environment has increased the global
public concern to seek alternative methods of
insect control. One of the promising alternatives is
the use of biological control methods, such as
biopesticides and entomopathogenic microorganisms. Entomopathogens (i.e. bacteria, fungi, and
viruses) are disease-causing organisms. They kill
or debilitate their host and are relatively specific to
certain insect groups. An important benefit of
biological control methods is that they can replace,
at least in part, some hazardous chemical pesticides
(Kumar et al., 2008). In addition, biological control
reduces expenses and health hazards associated
with pesticide sprays (Kouser and Qaim, 2011).
Bt is short for Bacillus thuringiensis, a natural
bacterium in the genus Bacillus. One of the best
modern agricultural defenses against plant-eating
insects is Bacillus thuringiensis (Bt), which either
can be applied to the surface of plants to provide
temporary protection or can be genetically engineered into the plant to protect against insects
throughout the lifespan of the plant (James, 2006).
The bacterial fermentation technology was led to
optimization of better formulations and cheaper Bt
products. New effective Bt species against insects
from the orders Lepidoptera, Diptera and Coleoptera were isolated (Cannon, 1995). With the
beginning of genetic engineering, genes for insect
resistance can be moved into plants more quickly
and deliberately (James, 2006).
Biotechnology affords opportunities to develop
new tools to treat pest and disease problems. When
a given pest or disease problem has no satisfactory
cure or treatment, usually only a technological
breakthrough can provide one, but the process
whereby this happens can be undefined and unpredictable. Bt technology is one example of genetic
engineering that may be used to develop insect
resistant crops now and in the future (Miller,
2007). However, the widespread success of Bt has
prompted two concerns; first, insects might
someday become resistant to this important treatment (Addison, 2010; Bates et al., 2005; Tabashnik
et al., 2009). Second, while primary pests are
controlled through Bt, the lower use of chemical
pesticides may entail the outbreak of secondary
pests, especially mirids, mealybugs, and other
sucking pest species, which are not controlled
through Bt (Lu et al., 2010; Nagrare et al., 2009).
Both factors could potentially lead to the increase
15
in using chemical pesticide again after a certain
time of reduction (Krishna and Qaim, 2012).
In 1996, the commercialization of transgenic Bt
maize (corn) hybrids to control corn rootworms
generated more interest and excitement among
corn growers who enthusiastically adopted the
technology to control the most important corn
pests in North America (Shelton et al., 2002). In
due course, transgenic traits to control both corn
borers and corn rootworms were “stacked” in elite
corn hybrids with traits for herbicide tolerance,
resulting in double, triple, and quad-stacked
hybrids (Gray et al., 2009). Next to Bt maize, Bt
cotton is currently the most widely grown Bt crop
in North America (James, 2010). The largest Bt
cotton areas are found in India and China, where
the technology is mainly used to control the
American bollworm Helicoverpa armigera and to
a lesser extent, spotted bollworm Earias vittella,
pink bollworm Pectinophora gossypiella, and
related species (Qaim, 2009), with efforts at
various stages in other countries to develop and
release adapted Bt-cotton varieties, such as South
Africa (Morse et al., 2004), Burkina Faso (Vitale et
al., 2010), Egypt (Dahi, 2012) and Kenya (Midega
et al., 2012).
Biological and Ecological Features of Bt
Bacillus thuringiensis (Bt) was first isolated about
112 years ago in Japan from silkworm larvae by
Ishwata in 1901. Ten years later, in Germany,
Berliner described the same pathogen from the
flour moth Ephestia kuehniella (Berliner, 1915).
For over 40 years, Bt has been applied to crops in
spray form as an insecticide, containing a mixture
of spores and the associated protein crystals. A
unique feature of Bt is that the bacterium produces
proteins in the form of crystalline structures, and
these proteins have activity against some insect
species (Riegler and Stauffer, 2003). Bt is naturally
occurring, gram positive, spore forming soil
bacterium (Figure 1), which differs from Bacillus
cereus in terms of its insect pathogenicity (Meadows, 1993). The characteristic of the parasporal
crystal, which is formed in the course of the
sporulation, was described early in 50’s (Hannay,
1953). Two years later correlated insecticidal activity with the parasporal crystal was reported
(Hannay and Fitz-James, 1955). The first sub-
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© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
species of Bt toxic to dipteran (flies) species was
found in 70’s (Goldberg and Margalit, 1977) and
the first discovery of strains toxic to species of
coleopteran was in 80’s (Krieg et al., 1986)
Figure 1: Spores and bipyramidal crystals of
Bacillus thuringiensis
This diverse genus also includes more than 20
other Bacillus species and hundreds of different
subspecies. Members of the genus Bacillus are
generally considered soil bacteria, and Bt is
common in terrestrial habitats including soil
(Martin and Travers, 1989; Meadows, 1993),
living and dead insects, insect feces, granaries, and
on the surfaces of plants. Bt occurs in nature
predominantly as spores that can disseminate
widely throughout the environment. Experiments
to isolate Bt from soil samples, which were treated
before with spore suspensions, have shown that
spores under natural conditions in the soil cannot
germinate and also do not outlast for a long period
of time (West et al., 1985). Other isolation attempts proved the fact that Bt is a ubiquitous
bacterium and is being found less in the soil than
on the plant parts, such as leaves and grain bran
from mills (Meadows et al., 1992; Smith and
Couche, 1991). A further source for Bt toxins
could be larvae, which died of bacterial infestations, for example, in beehives (Muerrle and Neumann, 2004).
Bt can synthesize a set of insecticide working substances: D-Endotoxin, A and B-Exotoxins, Phospholipases, immunosuppressive substances and
vegetative insecticide proteins (Krieg, 1986;
Peferoen, 1997). D-Endotoxin is the most important protein, which developed as crystal elimination during the sporulation, and it has a specific
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toxic effect against certain insect pests. In all
spores-forming bacteria, there is an exchange that
takes place between vegetative growth phase, with
intensive metabolism, and the following dwell
phase, as inactive spore formation. The vegetative
cells do not need special requirements of the
nutritive substrates and are therefore simply mass
produced. During exhaustion of nutrients, the cell
division is stopped and starts the sporulation
forming sporangium. This contains the actual
spore, surrounded by endo- and exosporium, as
well as parasporal protein crystals. Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal δ-Endotoxins (Cry toxins) which
are encoded by Cry genes. Cry toxins have specific
activities against species of the orders Lepidoptera
(moths and butterflies), Diptera (flies and mosquitoes) and Coleoptera (beetles) (Koziel et al.,
1993; Van Rie, 2000). Thus, B. thuringiensis
serves as an important reservoir of Cry toxins and
Cry genes for the production of biological insecticides and insect-resistant genetically modified
crops. After sporulation, the sporangium wall is
dissolved, and the spore and the parasporal body
are dismissed. Transgenic crops that contain Cry
genes are widely adopted by farmers in many
countries over the last 15 years (James, 2009).
Several studies showed that Bt crops, which
provide resistance to some Lepidopteran and
Coleopteran insect species, have helped reduce
chemical pesticide use and increase total yield
(Carpenter, 2010; Huang et al., 2005; Krishna and
Qaim, 2007; Morse et al., 2006; Qaim and De
Janvry, 2005; Subramanian and Qaim, 2009;
Wossink and Denaux, 2006) by 13-23% when
insect infestation was severe (Mungai et al., 2005),
but no significant differences were noticed in yield
under low or moderate insect infestation (Ma and
Subedi, 2005). Wang et al. (2010; 2012) found no
significant differences on growth performance or
total yield between Bt-transgenic rice and their non
Bt counterparts.
Krieg (1986) classified the pathotypes of Bt
species according to their effects on insect orders:
pathotype A; effective against butterflies, pathotype B; effective against Diptera and pathotype C;
effective against Coleoptera. Meanwhile, there are
other Pathotypes which are also effective against
other organisms like protozoan, mites and nematodes (Feitelson et al., 1992). Not all types of B.
thuringiensis bacteria synthesize crystalline pro-
International Journal of Agricultural and Food Research | Vol. 3 No. 1, pp. 14-40
teins that are toxic to insects (Bernhard et al.,
1997; Martin and Travers, 1989; Meadows et al.,
1992). Bt toxins increased mortality, reduced
growth rate, prolonged development time, and
reduced adult body mass and size of the insect
(Lang and Otto, 2010).
Structure of Bt D-Endotoxin
Bt D-Endotoxin is classified according to its
pathogenicity and the crystalline form into three
pathotypes (Schnepf and Whiteley, 1981). Crystalline proteins of the pathotype A have a bipyramidal form, proteins of the pathotype B are spheroid cuboids form and protein of the pathotype C is
rhomboid. In consequence of the discovery of new
toxin proteins and the inhomogeneous composition
of the protein crystals, a new nomenclature was
developed according to pathogenicity, sequence
homology and molecule-size (kDa) (Höfte and
Whiteley, 1989; Lereclus et al., 1993). A new
systematic procedure was developed by Peferoen
(1997) and Crickmore et al. (1998), which was
based only on the homology of the amino acid
sequence. Based on this, there were more than 100
well-known toxins, which were divided into 20
classes (Crickmore et al., 1998). The most studied
D-Endotoxins belong to the classes Cry1, Cry2,
Cry3, Cry4 (crystal protein) and Cyt (cytalase).
Table 1 is briefly describes crystal proteins, their
molecular weights and the target insects.
Table 1
Types of crystal proteins and the target insects:
D-Endotoxins
Crystal protein
Molecular
weight
Target insect
(active against)
Cry1
Cry2
Cry3
Cry4
Cyt
130 kDa
70 kDa
70 kDa
130 kDa
30 kDa
Butterflies (and beetles)
Butterflies (and Diptera)
Beetles
Diptera
Nonspecific Cytotoxin
Produced as crystalline inclusions by the bacterium
Bacillus thuringiensis (Bt), Cry toxins are the most
widely used insecticidal trait in transgenic crops
for insect control (James, 2009). Each crystal
protein consists of two terminals; amino terminal
and carboxyl terminal. During the activation of the
17
Cry protoxin, the carboxyl terminal is split by
proteases of the insect. The amino terminal
remains as active fragment of approximately 30
kDa (Cry4) - 60 kDa (Cry1), consisting of approximately 600 amino acids (Cry1). Cry2 and Cry3
toxins are hardly split (Gill et al., 1992), while the
Cyt toxin must not be activated (Lereclus et al.,
1993). By the means of the three-dimensional
analysis of the active Cry fragment, three different
domains were determined: Domain I; responsible
for the pores formation in the epithelium of the
midgut, domain II; interacts with the receptor
(Peferoen, 1997) and domain III; responsible for
the formation of a toxin oligomer (Pardo-Lopez et
al., 2006) that leads to osmotic cell death (Zhuang
et al., 2002).
Cry toxins target the insect midgut cells to compromise the gut epithelium barrier and facilitate the
onset of septicemia (Raymond et al., 2010; Soberon et al., 2009). Although the specific mechanism
resulting in enterocyte death is still controversial,
commonly accepted steps in the intoxication
process include solubilization of the crystal toxin
and activation by the insect gut fluids. Activated
toxins are attracted to the brush border membrane
of the midgut cells through low affinity binding to
glycosylphosphatidylinositol-anchored (GPI-) proteins (Arenas et al., 2010), such as aminopeptidaseN (APN) or membrane-bound alkaline phosphatase
(mALP). This initial binding step facilitates subsequent binding of higher affinity to cadherin-like
proteins (Bravo et al., 2004), which leads to further
processing of the toxin, resulting in formation of
toxin oligomers. Toxin oligomers display high
binding affinity towards N-acetylgalactosamine
(GalNAc) residues on GPI-anchored proteins
(Pardo-Lopez et al., 2006), resulting in concentration of toxin oligomers on specific membrane
regions called lipid rafts, where they insert into the
membrane, forming a pore that leads to osmotic
cell death (Zhuang et al., 2002). Alternatively,
binding of toxin monomers to cadherin has been
reported to activate intracellular signaling pathways that resulted in cell death by oncosis (Zhang
et al., 2006).
Bt Mode of Action
A unique feature of B. thuringiensis bacteria is the
synthesis of crystalline proteins, which have
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activity against some insect species. The main
effect of Bt against insects is due to D-Endotoxin.
Bt insecticides, whether in the form of spray or in
Bt-genetically engineered plants, do not function
on contact as most insecticides do. The insecticidal
effect of Bt comes from the crystal proteins (CPs)
produced during the bacterium’s sporulation phase.
These proteins are inactive in this phase. In order
to be activated, the crystals must be loosened in an
alkaline milieu (pH>9). The CPs can be converted
into the active toxin by protease during digestion,
which interacts with the epithelium of the midgut
(Riegler and Stauffer, 2003).
Among the agricultural pests that are currently
targeted with Bt insecticides are bollworms, stem
borers, budworms, and leaf worms in cereal crops;
the gypsy moth and spruce budworm in forests;
and the cabbage looper and diamondback moth in
vegetable crops (BCMAFF 2004; Olkowski et al.,
2000; Weinzierl et al., 2000). Mosquitoes and
black flies are also controlled with Bt sprays or by
treating the aquatic breeding sites with Bt (Lacey
and Merritt, 2004; Martin and Travers, 1989;
Peairs, 2010).
The CPs must be ingested by the target organism
to be effective. The process takes hours or even
days; somewhat longer time than that is required
for synthetic insecticides to kill insects. Active CP
binds to specific receptors on the midgut, forming
pores and leading to leakage of the midgut contents, paralysis and death of the insect. Recognition
of these toxins is necessary by receptors, which
locate on the microvilli of the midgut epithelium.
After perforation of the epithelial membrane by the
toxin, cations exchange increases between midgut
contents and the epithelial cells. As a result of the
increased osmotic pressure, the epithelial cells
burst and the midgut contents move into the
haemolymph causing blood poisoning and intestine
paralysis that results in insect death after few hours
to 2-7 days (Gill et al., 1992; Olkowski et al.,
2000; Riegler and Stauffer, 2003; Wheeler et al.,
2011). The pH of the insect gut (depending on how
alkaline or acidic it is) is critical for dissolving the
walls of the storage spore. Not all insects have the
same acidity in their gut, and this is why some
insects are susceptible to Bt poisoning (e.g., imamture stages of certain moths (caterpillars), beetles
(grubs), mosquitoes and black flies), while others
are not (BCMAFF, 2004; Olkowski et al., 2000;
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© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
Wheeler et al., 2011). The good thing is that these
reactions cannot take place in humans and other
mammals (Wheeler et al., 2011).
Bt Applications
1. Bt Preparations
For over 40 years, Bt has been applied to crops in
spray form as an insecticide. Bt preparations are
mostly suspensions that contain a mixture of
spores and associated protein crystals. Unlike other
pesticides that kill on contact, Bt must be eaten by
insects to be effective. As an ingredient of commercial sprays, Bt is relatively expensive compared
to chemical pesticides (Riegler and Stauffer, 2003).
Bt preparations are highly specific with short
persistence and thus have relatively high environmental compatibility. UV radiation breaks down Bt
and rain washes it off the plants. Therefore, Bt
must be applied exactly where and when the target
insects are feeding and they must consume it
quickly before it disappears (BCMAFF, 2004;
Martin and Travers, 1989).
The use of conventional Bt preparations is limited
against few insects. By the conjugation of different
Bt strains, a combination of different plasmids with
toxin genes in a specific strain can be made in
order to expand the host spectrum of target insects
(Cannon, 1995), for instance, most of the formulations found in retails indicate that they contain B.
thuringiensis var. kurstaki. This strain is limited to
controlling certain caterpillars. Other available
strains of Bt are effective treatments against larval
beetles and some flies, such as, incorporating B.
thuringiensis var. israelensis in the soil to kill
fungus gnats in greenhouse soil or in potted indoor
plants. Other strains, such as B. thuringiensis var.
san diego can be used to kill the Colorado potato
beetle (Olkowski et al., 2000). Bt preparations
degrade in short time after application, and this
short duration of effect forces to repeated spraying
(Behle et al., 1997).
Reliance on Bt in tree fruit production is also
rapidly increasing, as the United States environmental protection agency (EPA) restricts postbloom uses of higher-risk organophosphate (OP)
and carbamate insecticides. Innovative growers are
now perfecting insect integrated pest management
(IPM) programs by combining pheromone-based
mating disruption, Bt sprays, an application or two
International Journal of Agricultural and Food Research | Vol. 3 No. 1, pp. 14-40
of an insect growth regulator (IGR), and either
spinosad or a lower-risk OP or carbamate when
populations grow over thresholds (Benbrook and
Suppan, 2000). Pheromone-based mating disrupttion Bt sprays are now used in strawberry pest
management program in California (Devencenzi,
2013).
Rachel Carson promoted Bt as a natural insecticide
in her book, Silent Spring, published in 1962. By
the end of last century, the annual market extent of
Bt preparations world-wide was estimated by $60120 million (Cannon, 1995). Bt products was constituting about 90-95% of the total biopesticides
market (Feitelson et al., 1992), however, it shared
only 0.5-2% of the entire plant protection market
(Cannon, 1995; Mazier et al., 1997; Van Frankenhuyzen, 1993). By 1995, about 182 Bt-based
products were registered by EPA; however, by
1999 the total sales of Bt formulations constituted
less than 2% of the total sales of all insecticides
(EPA, 2010). Agro magazine points out that the
worldwide production of biopesticides in 2000 was
close to $160 million; of this, Bt biopesticides
accounted for more than 90%. Although the
worldwide pesticide market is in decline, under the
future IPM concept, it is predicted that the
biopesticides market will grow significantly. As of
the end of 2001, about 195 different biopesticides
effective ingredients and roughly 780 different
products have already been marketed. According
to Chuck Benbrook Consulting Company's forecast, after 2013, genetically modified crops and
biological additives will increase significantly by
7.5 times and 5 times, respectively (Yuan, 2010).
The global market for biopesticides was valued at
$1.3 billion in 2011 and is expected to reach $3.2
billion by 2017, growing at a compound annual
growth rate (CAGR) of 15.8% from 2012 to 2017.
North America dominated the global biopesticides
market, accounting for around 40% of the global
biopesticides demand in 2011. Europe is expected
to be the fastest growing market in the near future
owing to the stringent regulation for pesticides and
increasing demand from organic products (MAM,
2012).
2. Biotechnology of Bt Products
By the means of new molecular biology methods,
both the spectrum of use and the duration of effect
of Bt products can be expanded (Cannon, 1995;
Gelernter and Schwab, 1993). For example, by the
19
insertion of toxin crystals in other genetic systems,
for example, in non sporulated bacterium Pseudomonas fluorescens, the persistence of the toxin was
extended after application, since the toxin is
strongly more protected against dismantling processes (Schnepf et al., 1998). Further toxin genes
were inserted in bacteria, such as Clavibacter xyli,
in order to ensure a systemic protection against
stem borers, for example, in corn and sugar beet
(Turner et al., 1991). Transferring cry3Aa1 gene
into root nodules forming bacteria, such as
Rhizobium leguminosarum has protected legumenous roots against the larvae of Sitona flavescens
(Skot et al., 1990; 1994). Another possibility is the
transformation of insect baculovirus with Bt toxins,
in order to increase their virulence against certain
insect pests (Gelernter and Schwab, 1993; Vlak,
1995).
Reports on the emergence of insects resistance to
Bacillus thuringiensis D-Endotoxins have raised
doubts on the sustainability of Bt toxin-based pest
management technologies (Manyangarirwa et al.,
2006; Tabashnik et al., 2008), for instance, moth
Plutella xylostella has developed field resistance to
Bt toxin due to a reduction in toxin binding to gut
receptors (Ferre and Van Rie, 2002; Kain et al.,
2004; Shelton et al., 2002). Jurat-Fuentes et al.
(2011) reported that reduced levels of midgut
membrane-bound alkaline phosphatase (mALP) is
a common feature in strains of Cry-resistant Heliothis virescens, Helicoverpa armigera and Spodoptera frugiperda when compared to the susceptible
larvae. Insect resistance may also differ from one
cultivar of the insect to the other, for example,
there is evidence in the scientific literature that the
“variant” western corn rootworm is more difficult
to be killed with Bt proteins than the “normal”
western corn rootworm (Siegfried et al., 2005).
Since most of the insect-resistant transgenic plants
were released for commercial cultivation and the
target insect populations are consistently exposed
to the single Bt cry-toxin protein, therefore, the
possibility of insects evolving resistance to a single
Bt toxin is high (Gunning et al., 2005; Zhao et al.,
2005). This challenge has developed the
innovation of gene pyramiding that entails the
simultaneous expression of more than one toxin in
a transgenic plant (Manyangarirwa et al., 2006;
Shelton et al., 2002; Suresh and Malathi, 2013).
Theoretical and practical evidence in insect population genetics suggest that gene pyramiding
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20
cannot be sustained as a resistance management
strategy per se. Pyramiding is useful as a strategy
to broaden the range of insect pests controlled in
each transgenic variety, and it still has to be
deployed in tandem with Bt resistance management
strategies, such as crop refugia, biological pest
control, temporal and spatial crop rotations among
others (Manyangarirwa et al., 2006). Pyramided
transgenic chickpea plants with moderate expression levels of two Cry toxins showed high-level of
resistance and protection against pod borer larvae
of Helicoverpa armigera as compared to high level
expression of a single Cry toxin (Mehrotra et al.,
2011). The first two commercialized pyramided Bt
corn technologies in the U.S. for managing lepidopteran pests include Genuity VT Triple Pro and
Genuity Smart Stax. Both were first commercially
planted during the 2010 crop season (Monsanto,
2012; EPA, 2010).
3. Transgenic Bt-Plants
In May 1995, transgenic Cry3A potatoes were
certified for the first time in USA. One year later,
both Bt cotton and cry1Ab1 hybrid corn were also
certified (EPA 2000; Mazier et al., 1997). These
crops provide highly effective control of major
insect pests, such as the European corn borer,
southwestern corn borer, tobacco budworm, cotton
bollworm, pink bollworm, and Colorado potato
beetle and reduce reliance on conventional chemical pesticides. They have also provided notably
higher yields in cotton and corn (Betz et al., 2000).
In 1997, there were more than 3 million hectares
cultivated with transgenic Bt cotton, corn and
potatoes in the USA (EPA, 2000; Tabashnik et al.,
1997) with a potential for Bt genetically modified
crops to take up to 33% of the insecticide market
by 2000 (Arozzi and Koziel, 1997). Plantings of
Bt-maize (corn), expressing cry toxins harmful to
Lepidoptera or Coleoptera (Koziel et al., 1993;
Van Rie, 2000) grew from about 8 % of US corn
acreage in 1997 to 26 % in 1999, then fell to 19 %
in 2000 and 2001, before climbing to 29 % in 2003
and 67 % in 2012. Plantings of Bt cotton expanded
more rapidly from 15 % of US cotton acreage in
1997 to 37 % in 2001 and 77 % in 2012 (USDA,
2012).
The story started with the surprising discovery of
DNA interchangeability among different bacteria,
animals and plants makes it possible to locate the
gene that produces Bt proteins lethal to insects and
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© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
transfer this gene into crop plants. The plants
modified in this way are called transgenic. So,
plants can be genetically engineered to produce
their own Bt crystal protein (CP) which is toxic to
the pest species of concern. As the insect feeds on
the plant, it ingests the CP and suffers the same
fate as if it ingested leaf tissue sprayed with Bt
(Roush and Shelton, 1998). Transgenic plants
expressing insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt), are revolutionizing agriculture. Bt, which had limited use as a
foliar insecticide, has become a major insecticide
because genes that produce Bt toxins have been
engineered into major crops grown on 11.4 million
ha worldwide in 2000 and expanded to 80 million
ha in 2004. Constituting 19% of the worlds
genetically modified (GM) crops; these crops (i.e.
cotton, corn and potatoes) have shown positive
economic benefits to the growers and reduced the
use of other insecticides in a number of developed
and developing countries (Bates et al., 2005;
Cohen, 2005; Ferre and Van Rie, 2002; Ferry et
al., 2004; Gao et al., 2010; James, 2006; Shelton et
al., 2002; Wu and Guo, 2005). In United States,
corn farmers have experienced significant changes
since Bt corn was first introduced. Corn borer
infestations have decreased considerably; new
traits, such as corn rootworm and corn earworm
resistance, have been engineered into Bt seeds, and
many input costs have increased. Each of these
changes has the potential to alter the farm-level
costs and benefits of adopting and continuing to
plant Bt corn (Fernandez-Cornejo and Wechsler,
2012). The global area of genetically modified
crops is expected to increase significantly at 7.5
times after 2013 (Yuan, 2010).
The advantages of using transgenic Bt plants,
compared to the foliar sprays of Bt or chemical
pesticides, are: 1) Protection of the plant through
the entire vegetation period with no need of
repeated foliar sprays of Bt-biopesticides or other
chemical pesticides (BCMAFF, 2004), 2) Systemic
protection of plants against insects, for example,
stem borers, or resistant insects, which already
developed resistance against chemical insecticides
(Mazier et al., 1997). Bt is less likely than chemical
pesticides to cause field resistance in target insects
due to its short biological half-life and its
specificity (BCMAFF, 2004) and 3) Minimizing
the residues of chemical pesticides in food and
environment prevents the effects on non-target
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organisms and benefits farm workers and others
likely to come into contact with these pesticides
(James, 2000; NAS, 2000) because Bt toxins attack
sites are found only in target insects (Peairs, 2010).
Expression of Bt gene (cry1A) in tobacco and
tomato provided the first example of genetically
engineered plants for insect resistance (Barton et
al., 1987; Vaeck et al., 1987), which showed a
significant tolerance against Lepidopteran larvae
(Ely, 1993). Subsequently, several Bt genes have
been expressed in transgenic plants, including
tobacco, potato, tomato, cotton, brinjal, rice, and so
on (Kumar et al., 2008). In 1995, EPA registered
the first Bt potato-incorporated protectants (cry3A)
for use in the United States. Since then, EPA has
registered 11 Bt plant-incorporated protectants,
although 5 of these registrations are no longer
active and the rest are different cultivars of corn,
cotton and potato (EPA, 2002; 2011). By the cultivation of transgenic Bt cotton in America, the
application of insecticides reduced from 5-12 to 03 times, while in Australia about half of the insectcides amount was saved (Carpenter et al., 2002;
EPA, 2000; Roush, 1997). The largest Bt cotton
areas are found in India and China, where the
technology is mainly used to control the American
bollworm (Helicoverpa armigera) and to a lesser
extent, spotted bollworm Earias vittles, pink
bollworm Pectinophora gossypiella, and related
species (Qaim, 2009). In both countries, the cotton
sector is heavily dominated by smallholder farmers
with land areas of less than 5 ha, who benefit from
Bt technology adoption in terms of higher incomes
and lower occupational health hazards associated
with pesticide sprays (Hossain et al., 2004; Huang
et al., 2002; Kouser and Qaim, 2011; Qaim et al.,
2009). Bt cotton contributes to poverty reduction
and broader rural development in these countries
(Ali and Abdulai, 2010; Subramanian and Qaim,
2010). A unique panel survey of Bt cotton farmers
conducted in India between 2002 and 2008 shows
that the Bt pesticides reducing effect has been
sustainable. Total pesticides use has decreased
significantly over time and Bt has also reduced
pesticides applications by non-Bt farmers. Increase
in total yield ranged usually from 30 to 60% and
the reduction in number of insecticide sprays
averaged around 50% causing 50 to 110% increase
in profits from Bt cotton, equivalent to a range of
$76 to $250 per hectare (Choudhary and Gaur,
2010). Bt cotton biotechnology includes Bollgard I
21
technology, containing the Cry1Ac gene that was
officially commercialized in India in 2002 and
Bollgard II technology, containing stacked Cry1Ac
and Cry2Ab genes that was also approved in 2006
(Krishna and Qaim, 2012). By 2010, over six
million Indian farmers had adopted Bt cotton on
9.4 million hectares – almost 90% of the country’s
total cotton area (James, 2010). India is currently
the biggest producer of Bt cotton in the world since
2012 (Krishna and Qaim, 2012).
Outgoing from these transformation models, over
50 plants were successfully modified with the
toxin genes, cry1Aa1, cry1Ab1, cry1Ac1, cry1Ca1
and cry3Aa1 (Kumar et al., 2008). Transgenic
crops are grown over large areas in the America
(James, 2011; USDA, 2012). For 2012, US planted
area of wheat is estimated at 56.0 million acres,
soybean at 76.1 million acres, corn at 96.4 million
acres and cotton 12.6 million acres (NASS, 2012).
In Alabama alone, between 300,000 and 400,000
acres of Bt cotton has been grown annually since
1996. Syngenta Seeds Inc. reported development
of transgenic cotton plants expressing vip3Aa gene
across the US cotton-belt during 2000-2002. The
transgenic cotton plants were reported to provide
excellent protection against Lepidopteran insects
throughout the season and resulted in significantly
higher yields (Artim, 2003), however, in crops
such as cotton that are plagued by several pests
with varying degrees of susceptibility to Bt, there
is a concern that the toxins will not be strong
enough to kill all pests, because Bt toxins are
highly specific against insects without affecting
predators and other insects (Christou, 2005). The
result would be reduced Bt efficiency and
increased risk of pests developing Bt resistance
(Tabashnik and Carrier, 2010). Additionally,
reliance on a single (or similar) Bt protein(s) for
insect control increases the probability of Bt
resistance development in target pest populations
(Addison, 2010; Bates et al., 2005; Tabashnik et
al., 2009). Moreover, while primary pests are
controlled through Bt, the lower use of chemical
pesticides may entail the outbreak of secondary
pests, especially mirids, mealybugs, and other
sucking pest species, which are not controlled
through Bt (Lu et al., 2010; Nagrare et al., 2009).
Both factors could potentially lead to chemical
pesticide use increasing again after a certain time
of reduction. The probability of this happening
may be higher in the small farm sector of develop-
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22
ing countries, where implementation of Bt refuge
strategies and careful monitoring are more difficult
(Wang et al., 2008). However, beyond such
undesirable effects, there are also possible positive
spill-overs: widespread use of Bt technology may
suppress bollworm infestation levels regionally,
such that non-Bt adopters may also be able to
reduce their pesticide applications (Carrière et al.,
2003; Hutchinson et al., 2010; Wu et al., 2008).
Also, the insertion of toxin genes directly into the
chloroplasts DNA, which is similar to the bacterial
DNA, resulted in further chloroplasts is present in
large number (up to 5000) per cell. By specific
transformation of the chloroplast DNA, the toxin
part in the soluble protein content in the leaf can be
increased up to 3-5%, for instance, tobacco chloroplasts contain 5000-10,000 of their genome per cell
(McBride et al. 1995) with no native untransformed chloroplast genome without the toxin gene
present. This has established the homoplasmic
nature of transformed plants with massive number
of toxin gene per cell, explaining the high level of
tolerance to a specific insect in transgenic plants
with no effect on plant growth rate, physiological
processes or productivity (Daniell, 1999; 2000). In
addition, chloroplast genetic engineering offers a
number of other advantages as a plant-based expression system includes multi-genes engineering
in a single transformation event, lack of gene
silencing and position effects due to site specific
transgenic integration, minimal, or lack of pleiotropic effects due to subcellular compartmentalization of toxic transgene products, and transgene
containment via maternal inheritance (Daniell et
al., 2002; Maliga, 2004). Chloroplast transformation has been achieved in a much wider range of
dicot plant species, such as soybean, cotton, potato,
tomato, lettuce, sugar beet, eggplant, citrus etc.
(Verma and Daniell, 2007). Chloroplast transformation of monocots, including the most important food crops, such as rice, maize, wheat and
sorghum has not been successful yet (Clarke et al.,
2011). The levels of recombinant protein accumulation achieved by chloroplast transformation
vary enormously ranging from less than <1% to
more than 70% (Oey et al., 2009; Ruhlman et al.,
2010).
© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
Advantages and Disadvantages of Bt Plants
There are several advantages of expressing Bt
toxins in transgenic Bt crops;
1) The resistance is inherited in a stable and Mendelian fashion (Gould, 1988).
2) The level of toxin expression can be very high,
thus delivering sufficient dosage to the pest,
which is considered necessary to delay the
evolution of resistance (Bates et al., 2005).
3) Because the toxin expression is contained
within the plant system (in microgram quantities), the potential for exposure to farm
workers and nontarget organisms is extremely
low, and hence only those insects that feed on
the crop can be controlled effectively (Betz et
al., 2000), in addition, the toxin is highly
specific against insects without affecting predators and other beneficial insects (Christou,
2005; Kumar et al., 2008) because Bt toxins
attack sites are found only in target insects
(Peairs, 2010)
4) The toxin expression can be modulated by
using tissue-specific promoters that are found
in insect’s midgut, so Cry proteins are nontoxic to humans and pose no significant
concern for allergies. Food derived from Btprotected crops, which have been fully
approved by regulatory agencies, have been
shown to be substantially equivalent to the
food derived from conventional crops. Also,
the Cry proteins are rapidly degraded when
crop residue is incorporated into the soil or
plant products that were fed to animals or
human. Thus the environmental impact of
these crops is negligible (Betz et al., 2000;
Wheeler et al., 2011).
5) Bt-corn produces Cry1Ab protein throughout
the plant, which virtually eliminates corn
borer-induced tissue damage in corn products
making fungal spores less able to germinate
and produce fumonisins (fungal toxins) (Betz
et al., 2000). Fumonisins cause death and
morbidity in horses (Norred, 1993) and have
been linked in epidemiological studies to high
rates of esophageal and liver cancer in African
farmers (Marasas et al., 1988).
6) Reduced applications of broad spectrum insecticides (Carpenter et al., 2002; Edge et al.,
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International Journal of Agricultural and Food Research | Vol. 3 No. 1, pp. 14-40
2001; Krishna and Qaim, 2012; Romeis et al.,
2006), which save money and time (Morse et
al., 2004) and reduce health risks to farmers
and consumers (Kumar et al., 2008), in
addition to indirect positive impact on preserving beneficial insects populations and supplemental insect control by natural enemies (Betz
et al., 2000; Head et al., 2001).
Perceived disadvantages of Bt transgenic crops
may be:
1) Exchange of genetic material between the
transgenic crop and related plant species leading to the development of so called “Super
weed” (Kumar, 2002).
2) Increase in toxin levels in the soil may affect
soil microflora (Kumar, 2002; Wu et al.,
2004), however, this requires more research
because Wang et al. (2006) reported no
Cry1Ab protein in the rhizosphere soil of fieldgrown Bt transgenic rice.
3) Potential impact on nontarget species and the
environment (Kumar, 2002). Although the
transgenic Bt crop plants provide high levels of
protection against certain insect pests, they
may have consequent effects on natural enemies specializing on such pests (Ahl-Goy et
al., 1995). A better understanding of the interaction between transgenic plants, pests and
parasitoids is important to limit disruption of
biological control and to provide background
knowledge essential for applying measures for
the conservation of parasitoid populations. It is
also essential for investigation into the potential role of parasitoids in delaying the build-up
of Bt-resistant pest populations (Schuler et al.,
2003; Stewart et al., 2004). For example, by
feeding larvae of the European corn borer
Ostrinia nubilalis on transgenic Bt corn and
submitting it to the natural predator Chrysoperla carnea, doubled the mortality rate of this
predator (Hilbeck et al., 1998). Feeding the
wasp Meteorus laeviventris on Bt treated
larvae Ostrinia nubilalis increased the mortality and reduced the life span of the wasp
(Hafez et al., 1997). In USA, the cultivation of
transgenic Bt cotton has successfully reduced
infestation by both Pectinophora gossypiella
and Heliothis virescens, whereas Helicoverpa
zea caused substantial damage to the crop
(Roush, 1997). In other experiments with
23
transgenic Cry1Ab cotton in Australia the
infestation rates by Helicoverpa armigera were
reduced, however other pests, such as soft bugs
and Thripse increased, which made the application of synthetic chemicals necessary (Fitt et
al., 1994; Hardee and Bryan, 1997). Repeated
cultivation of transgenic Bt-plants may increase Bt toxins in the soil and cause changes
in the soil microbiology, for example research
on Bt corn, which includes Cry1Ab toxin,
revealed high mortality rate of the soil
collembola, Folosomia candida (Riegler and
Stauffer, 2003).
However, recent research findings negated the
effect of Bt-plants on non target species.
Ramirez-Romero et al. (2008) found that the
Bt maize expressing Cry1Ab toxin does not
affect the development of the non-target phytophagous aphid, Sitobion avenae on young
maize and that no presence of the toxin is
detected in this aphid species. They suggested
that there is no direct or mediated risk effect at
the third trophic level (parasitoids and
predators) associated with the aphid, Sitobion
avenae on Bt-maize. Li et al., (2011b) stated
that Bt cotton has no direct positive or negative
effect on a non-target pest Apolygus lucorum
in northern China and the observed outbreaks
in the insect numbers is due to the decrease in
the pesticide application. It was also reported
that Bt maize plants had no sub-chronic adverse effects on non-target Coleopteran insect;
Tenebrio molitor (Kim et al., 2012a) or Rhopalosiphum padi aphids (Kim et al., 2012b);
however, they stated that Cry toxins can be
transferred to higher predatory insects if they
consume the aphids feeding on Bt maize.
There would be a potential negative impact of
Bt maize expressing Cry3 in the food web in a
scenario where R. padi feeding on Bt maize
expressing Cry3 is consumed by a typical
Coleopteran predator, such as the lady beetle,
as Cry3 toxin is selectively toxic to Coleopteran insects. Thus, more detailed studies on
the potential impacts of the Bt toxin on the
food web are required.
4) Evolution of new and more virulent biotypes
of the pests because insects are capable of
developing high levels of resistance to one or
more Cry proteins (Kumar et al., 2008). As
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24
© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
such, the development of resistance in target
pests to Bt-plants is considered the main risk
for long-term success of this technology
(Farinós et al., 2011). So far, field evolved
resistance to Bt maize has been documented in
two species, the African stem borer, Busseola
fusca (Lepidoptera: Noctuidae) (Kruger et al.,
2009; Van Rensburg, 2007) and the fall
armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) (Matten et al., 2008; Storer
et al., 2010). In addition, laboratory selection
assays have shown that laboratory populations
of Ostrinia nubilalis (Lepidoptera: Crambidae)
can develop low to moderate levels of resistance under intense selection pressure from Bt
toxins (Crespo et al., 2009).
5) Reliance on one Cry protein may increases the
probability of Bt resistance development in
target pest (Addison, 2010; Tabashnik et al.,
2009), for example, pyramided Bt corn hybrids
were very effective, compared to single gene
Bt corn hybrids, against sugar cane borer,
Diatraea saccharalis (Ghimire et al., 2011;
Wangila et al., 2012), the dominant corn borer
species in many area of US gulf coast region
(Huang et al., 2012).
6) Fluctuations in toxin concentration occurs due
to various factors such as leaf age (Le et al.,
2007; Wei et al., 2005), growth conditions (Le
et al., 2007; Sachs et al., 1998), nutrient
availability (Coviella et al., 2002), Co2 level
(Chen et al., 2005; Coviella et al., 2002; Wu G.
et al., 2007) and elevated Co2; temperature and
tropospheric ozone (O3) could hasten Bt resistance in target insect (Himanen et al., 2009).
7) The toxin gene can be expressed in the
chloroplast genome and thus the possibility of
gene transfer via pollen (Daniell, 2000) is
highly variable (Szekacs et al., 2010). It was
reported that pollen from Bt corn is highly
toxic to Monarch butterflies in the laboratory
(losey et al., 1999), but follow up field studies
showed that under real-life conditions
Monarch butterfly caterpillars rarely come in
contact with pollen from corn (losey et al.,
1999; Sears et al., 2001). A similar conclusion
was reached for the pale grass blue butterfly,
Pseudozizeeria maha in Japan (Wolt et al.,
2005). Recently, Holst et al. (2013) reported a
potential environmental risk of the field
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cultivation of insect-resistant (Bt-toxin expressing) transgenic maize due to the consumption
of Bt-containing pollen by herbivorous larvae
of Inachis io butterflies (Lepidoptera) in
European farmland. In a more detailed analysis, they found that in northern Germany,
where Inachis io is mostly univoltine, the
cultivation of Bt maize would pose a negligible
risk, because pollen shedding is predicted later
than larval feeding, but in southern Germany,
where Inachis io is bivoltine, the second
generation of larvae coincides with the peak of
maize pollen deposition and consequently is at
risk. They concluded that the population-wide
effect of Bt maize on insect species will
depend on: the species-specific susceptibility
to Bt toxins; the actual exposure to the toxin,
and the ecology of the species. This conclusion
confirmed previous report by Peterson et al.
(2006) who modeled the phenology of both
maize pollen shedding and Karner blue
butterflies, Lycaeides melissa samuelis larval
feeding, and combined this with a GIS-based
analysis of the co-occurrence of Bt maize
fields and larval habitats, concluding that in
most places and for most years, maize pollen
shedding would occur after the majority of the
butterfly larva population had finished eating.
Bt-Resistant Pest Population
The potential of pest to develop resistance against
the defense mechanisms of crops is documented
and is not unique to genetically engineered plants
only. More than 500 insects and mites already have
acquired resistance to a number of insecticides
(McGaughey and Whalon, 1992) and similar
resistance to Bt toxins were developed in several
major pests, including the tobacco budworm, Colorado potato beetle, Indian mealy moth (Tabashnik
et al., 2003), maize stalk borer (Van Rensburg,
2007), cotton bollworm (Tabashnik et al., 2008)
and the fall armyworm (Storer et al., 2010). The
diamondback moth (Marois et al. 1991; McGaughey et al., 1998) and fall armyworm is
already known to have evolved a resistance to Bt in
spray form (Blanco et al., 2010), consequently loss
of the effectiveness of Bt preparations. This would
mean an enormous damage particularly for the
organic agriculture, where these preparations are
International Journal of Agricultural and Food Research | Vol. 3 No. 1, pp. 14-40
only certified as efficient Bio-pesticides (Madigan
and Martinko, 2005).
Due to constant exposure to the toxin, an evolutionary pressure is created for resistant pests and
the expression of the Bt gene can vary. For
instance, if the temperature is not ideal, this stress
can lower the toxin production and make the plant
more susceptible to insects attack. More importantly, reduced late-season expression of toxin has
been documented, possibly resulting from DNA
methylation of the promoter (Dong and Li, 2007;
Müller-Cohn et al., 1996). Bt-toxin resistance
evolution in herbivore insects has been raised as a
severe threat for the continuing success of Bttransgenic crops (Tabashnik et al., 2008).
There is also another risk that, for example,
transgenic maize will crossbreed with wild grass
variants, and that the Bt-gene will end up in a
natural environment, retaining its toxicity. An
event like this would have ecological implications
(Wu et al., 2004), as well as increasing the risk of
Bt resistance arising in the general herbivore
population (Kumar, 2002). The frequency of resistance alleles in Helicoverpa zea to Bt Cry1Ac
cotton have been reported to increase in field populations, but resistance management tactics, such as
refuge requirements (Frisvolda and Reeves, 2008)
and concurrent expression of several toxins in the
same plant (Ibargutxi et al., 2008) have been
successful in delaying the onset of resistance
(Tabashnik et al., 2008), especially that insectspecific CPs cannot be changed during a growing
season (Habuštová et al., 2012; Webber 2006).
Resistance Management
There are many methods for delaying the
appearance of resistant strains of the insect to Bt
crops. One of the key factors for successful
resistance management is the timely implementation of monitoring programs to detect early
changes of susceptibility in the field populations in
relation to the original susceptibility of the population (Farinós et al., 2004), jointly with the implementation of resistance management strategies to
prevent or delay the emergence of resistant populations. Different strategies have been proposed to
delay insect resistance to Bt crops, but the “high
dose/refuge strategy” (HDR) is the most widely
used, and it is mandatory in most countries where
25
Bt crops are grown (Farinós et al., 2011). This
strategy combines the use of Bt crops that express
high concentrations of Cry toxins and the planting
of refuges of non-Bt crops near Bt crops. The aim
is to encourage a large population of pests so that
any genes for resistance are greatly diluted, and
thus reduction of resistance heritability. This technique is based on the assumption that resistance
genes will be recessive (Bravo and Soberón, 2008;
Blanco et al., 2010; Carrière et al., 2010; Liu and
Tabashnik, 1997; Storer et al., 2010; Tabashnik,
1994; Tabashnik et al., 2004). Gao et al. (2010)
stated that the resistant alleles will be diluted by
susceptible moths produced from surrounding
areas, which may be a major factor contributing to
maintenance of susceptibility in this species to
Cry1Ac after a decade of large-scale planting of Bt
cotton. Li et al. (2011a) found that based on the
relative average development rates (RADR) of H.
armigera larvae in F1 generation, no substantial
increase in Cry1Ac tolerance was found over the 3years period. It appears so far to be a successful
method of delaying widespread resistance to Bt
toxins (Carrière and Tabashnik, 2001; Frisvolda
and Reeves, 2008; McGaughey et al., 1998; Tabashnik et al., 2003). Increasing Bt expression levels
(high dose/structured refuge strategy), which has
been adopted for planting the first generation Bt
corn that expresses a single Bt protein (e.g. Yield
Gard Bt corn), is based on the assumptions that
resistance in the target species should be recessive
so that a high percentage of resistant heterozygotes
can be killed by “high dose” expressed Bt corn
(Andow and Hutchison, 1998; EPA, 2001); however, some recent findings reported that single
gene Bt corn did not express a high dose of Bt
protein, as desired for the high dose/refuge strategy
(Ghimire et al., 2011; Wu X. et al., 2007).
Alternately, creating a mosaic genetically modified
(GM) crop expressing many different Bt toxins
would have a greater chance of eliminating the
entire pest population and thus eliminating resistance alleles (Atkinson, 2006; Ibargutxi et al.,
2008). Expressing multiple toxins, that is, gene
pyramiding, which is a strategy employed to
develop transgenic plants that express that multiple
Bt proteins is more effective than one protein in
targeting the same group of insect pests (Manyangarirwa et al., 2006; Monsanto, 2012; Shelton et
al., 2002; Suresh and Malathi, 2013; Wangila et
al., 2012). Because CrylAb and CrylAc are very
Science Target Inc. www.sciencetarget.com
26
similar in their structure and function, resistance to
one CrylAb protein would most likely impart
resistance to another CrylAc protein as has already
been observed with the tobacco budworm. Nowhere is this more of a concern than with cotton
bollworm/corn earworm that usually feeds on corn
during spring and early summer, then migrates to
cotton to complete several more generations during
summer and early fall (Wearing and Hokkanen,
1995). Clearly, different Bt proteins are needed to
decrease the development of resistance (Atkinson
2006; Ibargutxi et al., 2008).), for example pyramided Bt corn hybrids were very effective, compared to single gene Bt corn hybrids, against sugar
cane borer, Diatraea saccharalis (Ghimire et al.,
2011; Wangila et al., 2012), the dominant corn
borer species in many area of US gulf coast region
(Huang et al., 2012).
Another method is expressing the protein only in
tissues highly sensitive to damage (tissue specific
expression) (Fearing et al., 1997; McGaughey and
Whalon, 1992). By means of spatial and temporal
regulation of toxin expression, DNA- technology is
offering promising solutions to minimize the
residues of Bt toxins in soil and delaying build-up
resistant strains of insect pests. For example, by
expressing insecticidal proteins in chloroplasts,
toxins can be put in the part of a plant where they
are most likely to be consumed. The toxin gene can
be expressed in the chloroplast genome and thus
the possibility of gene transfer via pollen (Daniell,
2000), in which toxin concentration is highly
variable (Szekacs et al., 2010). Most caterpillars
feed on green tissues that are rich in chloroplasts;
therefore, they consume the highest level of insecticidal toxins if the toxins are placed in chloroplasts. Related studies indicated that high levels of
expression of Ccry2Aa2 in transgenic tobacco did
not affect growth rates, photosynthesis, chlorophyll
content, flowering, or seed forming under laboratory conditions (EPA, 1995; Fearing et al., 1997).
Gómez-Barbero et al. (2008) reported a significant
yield advantage of Bt maize over conventional
maize. Incorporating these genes in the chloroplast
offers several advantages, including the ability to
place foreign genes at a specific location in the
plant cell and to increase the levels of toxic
proteins in the plants. Furthermore, because chloroplast genes are inherited through the mother
(ovary) instead of the father (pollen), the risk for
out-crossing (foreign genes escaping to other
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© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
species) to other plants, such as weeds, is reduced
(Mikkelsen et al., 1996). Gene transfer between
cultivated plants and wild species is well-known.
By out-crossing or introgression, the inserted genes
of transgenic plants can be transferred over pollen
to the same species of cultivated plants and wild
species. The out-crossing rate is however different
depending upon plant type and geographical
location and must be regarded (Pascher and
Gollmann, 1997). There are many examples such
as strawberries, carrot, corn, sorghum, sunflowers
and sugar beet where out-crossing is conceivable
into other species (Gray and Raybould 1998; Kling
1996). Stewart et al. (1997) enumerates nine
species of Brassica napus, to which an out-crossing is possible within short time.
Horizontal gene transfer simply means spreading
of genes between very different species in non
sexual ways. Moreover, it takes place via transduction, transformation, conjugation, as they arise
frequently with bacteria, as well as via the activity
from transposing, retroviral and other infectious
agents. Thus, it differs from the vertical gene
transfer of vegetative and sexual form inclusive
species hybridizing and introgression. Like that
some sequence homologue between bacteria and
plants are well-known, which suggest a gene
transfer in the course of the evolution. A possible
gene transfer of Bt toxin genes on related Bacillus
sp. is also conceivable (Schlüter and Potrykus,
1996). During the risk estimation of transgenic
plants, a possible horizontal gene transfer should
be considered primarily by plants to soil bacteria
and mushrooms (Lorenz and Wackernagel, 1994).
A restriction of the out-crossing can take place via
transformation of the chloroplast. The propagation
of the transgenes over pollen is thus prevented,
since plastids are left purely maternal (Daniell et
al., 1998; Schlüter and Potrykus, 1996). McBride
et al. (1995) reported that there is a high level of
expression of Cry2Aa2 in tobacco chloroplasts
compared to Bt genes inserted into plant nuclei.
This is likely because DNA can be expressed better
in chloroplasts than in nuclei, and there are many
chloroplasts within a plant cell, but only one
nucleus. DNA placed in the chloroplasts will be
copied 5,000 to 10,000 times in a cell, while
typically there are only one to four copies of the
gene per cell when it is contained within the
nucleus. Also, plant cells can express smaller
genes (Cry2Aa2) better than larger (CcrylAc) genes
International Journal of Agricultural and Food Research | Vol. 3 No. 1, pp. 14-40
(Daniell et al., 1998; Feitelson et al., 1992; Fitt et
al., 1994).
Conclusion
Bacillus thuringiensis (Bt) is insect-pathogen
bacteria, its impact is mainly due to the synthesis
of D-Endotoxin. This diverse genus also includes
more than 20 other Bacillus species and hundreds
of different subspecies. Members of the genus
Bacillus are generally considered soil bacteria, and
Bt is common in terrestrial habitats including soil,
living and dead insects, insect feces, granaries, and
on the surfaces of plants. When a susceptible host,
eats the crystal, part of it binds to specific gutreceptors, penetrates, and collapses the cells lining
its gut, causing death.
For decades, produced Bt preparations consisting
of spores and toxins are registered as Bio-pesticides. Advantages of the Bt preparations are high
specificity, short persistence and thus a relatively
high environmental compatibility. UV radiation
breaks down Bt and rain washes it from the plants.
Therefore, Bt must be applied exactly where and
when the target insects are feeding and they must
consume it quickly before it disappears. The use of
conventional Bt preparations is limited against few
insect pests. By the conjugation of different Bt
strains, different plasmids can be combined with
toxin genes in a single strain, in order to expand
the host spectrum of targeted insects. Bt preparations degrade in a short time after application and
this short duration of effect forces repeated spraying. Bt in spray form is environment friendly and
considered as certified efficient Bio-pesticide for
organic agriculture.
Plants can be genetically engineered to produce
their own Bt crystal protein, which is toxic to the
pest species of concern. As the insect feeds on the
plant, it ingests the crystal protein and suffers the
same fate as if it ingested leaf tissue sprayed with
Bt (Roush and Shelton, 1998). The use of commercial crops expressing Bt toxins has increased in
recent years due to their advantages over crops that
require traditional chemical insecticides.
27
Some advantages to the use of transgenic Bt plants
compared with foliar sprays of Bt are as follows:
protection of the plant through the entire vegetation period; minimizing the residues and the side
effects of pesticides in the environment, and systemic protection of plants against insects, for
example, stem borers, or resistant insects, which
already developed resistance against chemical
insecticides. On the other hand, due to the constant
exposure to the toxin an evolutionary pressure is
created for resistant pests, and hence the expression of the Bt gene can vary. The potential of pests
to develop resistance against the defense mechanisms of crops is well-known, and is not unique to
genetically engineered plants. Because more than
500 insects and mites already have acquired resistance to a number of insecticides, there is concern
that similar resistance to Bt toxins could develop.
In addition, although the transgenic Bt crop plants
provide high levels of protection against certain
insect pests, their consequent effects on non target
species is still controversial, in spite of recent
findings that proof that there are little or no effect
on non target species.
Within the next few years, crops that have been
genetically engineered for Bt resistance could
dramatically lower production costs and provide
farmers with new insect control options. The
success of their commercialization depends on
several factors, including the regulatory climate,
patent issues, and the ability of scientists to deal
with targeted insects that develop resistance to
lethal proteins. Before a Bt plant is released, and
especially before it is authorized for commercial
cultivation, tests have to be carried out to check
that this plant will not be associated with any
harmful impacts on non-target organisms. In
addition, they have to reach a result within a
reasonable amount of time, as well as take into
account complex ecological relationships.
Hence, it can be concluded that using of transgenic
Bt-plants is so far promising for the future of crop
protection, however, some cautions, related to the
unknown future effects on human health and other
organisms, should be taken into account and more
research conducted to proof that.
Science Target Inc. www.sciencetarget.com
28
© Ibrahim and Shawer 2014 | Transgenic Bt-Plants
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